COMMERCIAL
FISHING
ENVIROMENTAL
POLLUTION
ASSESSMENT
RESTOCKING OF
NATURAL HABITATS
JUVENILES FROM
AQUACULTURE
SPORT
FISHING
COMMERCIAL
AQUACULTURE
DIGESTIVE
ENZYMES
AQUARIUM
TRADE
BYPRODUCTS
UTILIZATION
HANDYCRAFTS
&
TROPHEES
• Native species from the
Atlantic coast of North
America
• Top position in the food
web and occasional
scavenger
• Availability of individuals
raised under controlled
conditions
• High metabolic rate (e.g.
growth)
• Able to tolerate different
physicochemical
conditions (e.g.saltwater,
O2, ammonia, etc)
• Continuous contact with
the bottom
• Ancient fish (simple
metabolic pathways)
Establishment of lab and field
methodologies to asses the impact of
aquatic pollution by means of
biomarkers in Alligator gar (A. spatula)
1) BIOMARKERS SELECTION:
 Implementing lab techniques for the
evaluation of different biomarkers
 Determining the response of alligator gar,
of different ages to known environmental
disruptors
1) Esterases
• Carboxylesterases
• Acetyl cholinesterase
• Butiryl cholinesterase
1) Detoxification
Cytochrome P450
(CYP1A)
2) Detoxification
Glutathione S-transferase
2) Endocrine Disruption
Vitellogenin
(VTG)
3) Oxidative stress
Superoxide Dismutase
4) Tissue injury
Alkaline phosphatase
Alligator gar: 3/Treatments
1) Hydrocarbure (H)
1 year: 60 g; 23 cm
b-naphtophlavone
2 years: 309 g; 38 cm
2) Pesticide (P)
Treatments application
Organophosphorate
50 mg/1000g in fish oil
(Diazinon)
Intraperitoneal injection
3) Endocrine Disruptor (ED)
(exposure: 48 h)
17b-Estradiol
Tissue samples:
4) Control (C)
Liver (L); Plasma (P);
Fish Oil
Gills (G); Mucus (M)
(Menhaden Oil)
ALLIGATOR GAR
F
I
L
T
E
R
500 L
Fiber
glass
tanks
Biomarker
Determination
Treatment application
(50 mg / 1000 g)
Tissue
sample
Sample in
liquid N2
•A group of isozymes mainly located in RER
•Involved in metabolism of a wide variety of xenobiotics
containing esther, thioesther and amide groups.
•Highest affinity for pesticides than related family of
cholinesterases (acetyl and butiryl)
•Pesticides cause activity inhibition in most tissues
• e.g. Liver
•Other xenobiotics cause Increase of activity
ASSAY METHOD
Enzyme kinetics in 96-well microtiter plate using
p-nitrophenyl acetate as substrate (Munilla & Starck, 1990)
0.02 ml
0.2 ml
substrate
0.02 ml
Buffer
Plus
Abs.
405 nm
10 min.
Enzyme
extract
Carboxylesterase activity in 1 year alligator gar
0.6
A
Abs/min/mg protein
0.5
0.4
B
B
L
G
0.3
0.2
0.1
A
A
A
0
ED
H
Treatments
C
Carboxylesterase activity in 2 years alligator gar
0.6
A
A
A
Abs/min/mg protein
0.5
0.4
B
0.3
A
A
B
AB
0.2
0.1
A
A
A
A
0
ED
H
C
Treatments
P
P
L
G
•Nonspecific phosphomono-esterase that removes a
phosphate group and transfers it to other molecules.
•Pollutants cause activity variation in several tissues.
•Increased activity in the plasma in a variety of disease
conditions.
•Biomarker of tissue injury.
ASSAY METHOD
Enzyme kinetics in 96-well microtiter plate using
4-nitrophenyl phosphate as substrate (Moyano et al , 1996)
0.02 ml
0.2 ml
substrate
0.02 ml
Buffer
Plus
Abs.
405 nm
10 min.
Enzyme
extract
Alkaline phosphatase activity in 1 year alligator gar
1.2
A
Abs/min/mg protein
1
B
0.8
B
L
G
0.6
0.4
0.2
A
A
A
0
ED
H
Treatments
C
Alkaline phosphatase activity in 2 years alligator gar
1.6
A
Abs/min/mg protein
1.4
1.2
AB
A
B
1
A
A
0.8
L
G
A
B
0.6
0.4
0.2
0
ED
H
C
Treatments
P
•Vtg is an estrogen-inducible protein synthesized by the
liver.
•Males do not produce Vtg in measurable quantities
under normal conditions, however have the capacity to
synthesize it.
•Many chemicals that are released into the environment
possess estrogenic properties.
•Measurement of Vtg in males and juveniles provides an
excellent biomarker for estrogenic compounds.
VTG
VTG Alligator
gar VTG
VTG
VTG
defatted
milk
VTG
VTG
Incubation
immobilization
washing
VTG
Saturation
washing
VTG
VTG
+
VTG VTG VTG
VTG
Competition
washing
Secondary
antibody
METHOD:
Homologus competitive
ELISA
(Mendoza et al., In Press)
VTG
VTG
VTG
washing
incubation
enzyme
substrate
OPD
VTG
VTG
VTG
VTG
Standar
Vtg or
Sample
anti-VTG
Optical
density
measure
ng de vtg/mg protein
VTG concentration in 1 year Alligator gar
100
90
80
70
60
50
40
30
20
10
0
A
L
G
M
A
A
ED
B
B
B
B
H
C
Treatments
VTG concentration in 2 years alligator gar
ng vtg/mg protein
120
A
100
80
P
L
60
G
B
40
B
M
B
20
0
ED
H
C
Treatments
P
Environmental disruptor
Biomarker
Hydrocarbure
Carboxyl esterase
Alkaline
phosphatase
Vitellogenin
Slight Reduction
(1 year)
Slight Reduction
Pesticide
Estrogenic
Reduction Increase
Increase
Increase
Increase